Mitochondrial energy metabolism in a model of
undernutrition induced by dexamethasone.
Jean-François Dumas, Gilles Simard, Damien Roussel, Olivier Douay,
Françoise Foussard, Yves Malthiery, Patrick Ritz
To cite this version:
Jean-François Dumas, Gilles Simard, Damien Roussel, Olivier Douay, Françoise Foussard, et
al.. Mitochondrial energy metabolism in a model of undernutrition induced by dexamethasone..
Br J Nutr, 2003, 90 (5), pp.969-77. <inserm-00128557>
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Br J Nutr 11/2003; 90(5): 969-77
HAL author manuscript
Mitochondrial energy metabolism in an experimental dexamethasone malnutrition
model.
Jean-François Dumas, Gilles Simard, Damien Roussel, Olivier Douay, Françoise Foussard,
Yves Malthiery and Patrick Ritz*
inserm-00128557, version 1
INSERM EMI-U 00.18, Laboratoire de Biochimie et de Biologie Moléculaire.
*For correspondence, Medecine B, CHU, F-49033 ANGERS Cedex 01
Phone: +33 241354499, fax : +33 241354969, e-mail : [email protected]
Short title: Mitochondrial energetics in DEX-treated rats.
Keywords: glucocorticoid, mitochondrion, oxidative phosphorylation, and respiratory chain
complexes.
Abstract
This investigation was undertaken to evaluate whether mitochondrial energy metabolism is
altered in a malnutrition model associated with dexamethasone treatment (1.5mg/kg/day for 5
HAL author manuscript
days). Gastrocnemius and liver mitochondria were isolated from dexamethasone (DEX)treated, pair-fed (PF) and control (CON) rats. Body weight was significantly more reduced in
DEX-treated group (-16%) than in PF group (-9%). Dexamethasone increased the liver mass
(+59% vs. PF and +23% vs. CON) and decreased gastrocnemius mass. Moreover, in DEX-
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treated rats, liver mitochondria exhibited an increased rate of non-phosphorylative oxygen
consumption with all substrates (approximately +42%). There was no difference in enzymatic
complex activities in liver mitochondria between rat groups. Collectively, these results
suggest an increased proton leak and/or redox slipping in liver mitochondria of DEX-treated
rats. In addition, dexamethasone decreased the thermodynamic coupling and efficiency of
oxidative phosphorylation. We therefore suggest that this increase in the proton leak and/or of
redox slip in liver is responsible for the decrease in the thermodynamic efficiency of energy
conversion. In contrast, none of the determined parameters of energy metabolism were altered
by dexamethasone in gastrocnemius mitochondria. Therefore, it appears that dexamethasone
specifically affects mitochondrial energy metabolism in liver.
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1
Introduction
2
Malnutrition is highly prevalent among patients, especially elderly people. It is responsible for an
3
increased mortality, significant hospital expenditure, reduced muscle and immune function, and
4
decreased quality of life (Wallace et al. 1995; Tucker & Miguel, 1996; Chima et al. 1997; Lesourd
5
& Mazzari, 1997; Landi et al. 2000). Malnutrition results from a negative energy balance, a
6
situation where energy intake fails to meet energy requirements. Although anorexia and a reduced
7
energy intake are always associated with malnutrition, in some clinical circumstances, an increased
8
resting oxygen consumption rate (referred to as an increased energy requirements) can be shown
9
(Nguyen et al. 1999). On the contrary, most human and non-human studies show that energy
10
restriction decreases energy expenditure (Ramsey et al. 2000). Therefore it appears that in these
11
clinical states, adaptive mechanisms which lead to a reduction of energy requirements, fail to
12
operate. However, the biochemical nature of this negative energy balance phenomenon is poorly
13
understood at the present time. A wasting of energy may be a possible explanation. In other words,
14
oxidative phosphorylation may be less efficient.
15
High-dose glucocorticoid treatment affects body weight and body composition (Kochakian
16
& Robertson, 1951; Hausberger & Hausberger, 1958). It has also been found to induce a
17
hypercatabolic state which leads to a reduced muscle mass (Marone et al. 1994; Minet-Quinard et
18
al. 1999), suppressed protein synthesis, a transient increase in protein degradation and a negative
19
nitrogen balance (Bowes et al. 1996; Max et al. 1988; Odedra et al. 1983). Moreover, it is a model
20
of hypercortisolism, which occurs during metabolic stress in humans and which is associated with
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an increased energy expenditure (Woodward & Emery, 1989; Brillon et al. 1995; Tataranni et al.
22
1996). On the other hand, acute high-dose treatment (<1 week) of rats with dexamethasone
23
decreases food intake (Kaur et al. 1989; Minet-Quinard et al. 1999). Therefore, a negative energy
24
balance ensues, both because of an increased energy expenditure and a decreased energy intake.
25
The mechanisms leading to this increased energy expenditure are not fully understood. There are
26
arguments to suggest that mitochondrial energy production could be affected. However, the effects
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of glucocorticoids on cellular energy metabolism depend on the tissues being investigated and on
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the duration of treatment. In the liver for example, short-term administration (<24 hours) of
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dexamethasone appears to increase oxidative phosphorylation while having no effect on non-
30
phosphorylative respiration (Wakat & Haynes, 1977; Allan et al. 1983). On the contrary, longer
31
administration (<1 week) of glucocorticoids, results in decreased liver oxidative phosphorylation
32
and ATP synthesis when fuelled through complex I, however remaining unchanged when fuelled
33
through complex II or IV (Kerppola, 1960; Kimura & Rasmussen, 1977; Jani et al. 1991). In
34
isolated skeletal muscle mitochondria, studies show either no change, a decrease or an increase in
35
oxidative capacity (Cytochrome c oxidase activity) or in oxidative phosphorylation (Vignos &
36
Greene, 1973; Koski et al. 1974; Capaccio et al. 1985; Marone et al. 1994; Weber et al., 2002).
37
Since liver and muscle contribute approximately 50% of body oxygen consumption (Rolfe
38
& Brown, 1997), most of it being coupled with ATP synthesis, we undertook this study to
39
investigate the effects of 5 days of high dose (1.5mg/kg/day) dexamethasone on energy metabolism
40
particularly in muscle and liver mitochondria. The comparison with caloric restriction helps in the
41
understanding of the increased energy expenditure observed at the whole body level.
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Materials and methods
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Animals
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The present investigation was performed in accordance with the French guiding principles in the
45
care and use of animals. Thirty-two male Sprague-Dawley rats, born and bred in our animal facility,
46
were housed in individual cages at 9 weeks of age (300-350g). Animals were provided with water
47
ad libitum and a standard diet (U.A.R A04) consisting (% weight) of 16% protein, 3% fat, 60%
48
carbohydrate and 21% water, fibre, vitamins and minerals. The metabolizable energy content was
49
12 kJ/g. Rats were divided into 4 groups of 8 as follows: dexamethasone(DEX)-treated rats received
50
a daily intraperitoneal injection of 1.5 mg/kg of dexamethasone for 5 days. Due to the fact that
51
dexamethasone treatment induces anorexia, pair-fed (PF) rats were used to discriminate between the
52
effect of anorexia and the effect of dexamethasone itself on the parameters measured. PF rats were
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pair-fed with DEX-treated animals (rats received the same food quantity consumed by DEX-treated
54
rats the previous day) and were injected daily with an isovolumic solution of 0.9% NaCl. Rats from
55
the control group (CON) were healthy, received no treatment, and were fed ad libitum. Animals of
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the control injected group (CI) were fed ad libitum and were injected with an isovolumic solution of
57
0.9% NaCl. This group was used to study the effects of NaCl injection. As results were similar
58
between the two control groups, the CI group was omitted in the presentation of data. Experiments
59
were conducted over a 5-day period. The dose and duration of the dexamethasone treatment was
60
chosen with reference to the literature and is known to induce a reproducible maximum
61
hypercatabolic state (Minet-Quinard et al. 2000). On the 4th day, following an overnight fast, the
62
animals were killed by decapitation. Gastrocnemius, liver and interscapular brown adipose tissue
63
were removed rapidly and weighed. Some tissue samples were immediately used for respiratory
64
measurements and the remainder were frozen in liquid nitrogen and stored at -80°C in order to
65
measure enzyme activity levels. Gastrocnemius muscle was chosen because this mixed-fibers tissue
66
is representative of muscle fiber types contained in the hindlimb of the Sprague-Dawley rat
67
(Armstrong & Phelps, 1984).
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Mitochondrial enzyme activities
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Frozen liver and gastrocnemius (10-30 mg) were thawed and homogenized with a Potter-Elvehjem
71
homogeniser (7 strokes) in an isolation medium consisting of 220 mM mannitol, 75 mM sucrose, 10
72
mM Tris and 1 mM EGTA, pH 7.2. Each homogenate was centrifuged at 600 g for 10 min and the
73
resulting supernatants were filtered through cheesecloth. All procedures were performed at 4°C.
74
The activity of citrate synthase, succinate dehydrogenase and complexes I, III and IV was measured
75
spectrophotometrically at 37°C in the supernatant fraction via an adaptation of that as described by
76
Malgat et al. (1999), and in agreement with the Mitochondrial Diseases Group of the Association
77
Française de Myopathie (AFM). Protein concentration was determined using the Bicinchoninic acid
78
Assay kit (Interchim, Montluçon, France) with bovine serum albumin (BSA) used as a control.
79
The activity of citrate synthase (CS) was measured in a reaction medium consisting of 100
80
mM Tris/HCl, 40 µg/ml 5,5'-dithio-bis(2-nitrobenzoic acid), 1 mM oxaloacetate, 0.3 mM acetyl
81
CoA and 4 % of Triton X 100, pH 8.1. After 3 min of incubation, the reaction was initiated by
82
adding the homogenate (20 to 50 µg proteins) and the change in optical density at 412 nm was
83
recorded for 3 min.
84
The activity of succinate dehydrogenase was measured by following the reduction of 2,6-
85
dichlorophenolindophenol (DCPIP), in the presence of phenazine methosulfate (PMS) at 600 nm.
86
Homogenate (20 to 50 µg proteins) was preincubated in a buffer containing 50 mM KH2PO4, 16
87
mM succinate, 1.5 mM KCN, 100 µM PMS, pH 7.5 for 5 min. The reaction began with the addition
88
of 103 µM DCPIP and the optical density was recorded for 3 min.
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The activity of complex I was determined by monitoring the oxidation of NADH at 340 nm.
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Homogenate (40 to 100 µg proteins) was preincubated for 3 min in 820 µl of distilled water.
91
Following this, 10 mM KH2PO4, 2 mM KCN, 5 µg/ml antimycin A, 100 µM decylubiquinone, 1.3
92
mg/ml BSA, 5 mM MgCl2, pH 7.5 were added. The reaction was initiated by the addition of 200
93
µM NADH and the change in the optical density was analysed for 3 min. The NADH
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decylubiquinone reductase activity was also measured in the presence of 12.7 µM rotenone. The
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specific activity of complex I represented the difference between NADH oxidation activity, both
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with and without the rotenone.
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The activity of complex III was determined by monitoring the reduction of cytochrome c at
98
550 nm. Homogenate (20 to 50 µg proteins) was incubated for 30 sec in a reaction medium
99
consisting of 35 mM KH2PO4, 5 mM MgCl2, 2.5 mg/ml BSA, 1.8 mM KCN, 125 µM oxidized
100
cytochrome c, 12.5 µM rotenone and 62.5 mM EDTA, pH 7.5. The reaction was initiated by adding
101
80 µM decylubiquinol and the optical density was measured for 3 min. The nonenzymatic reduction
102
of cytochrome c was measured under identical conditions after the addition of 10 µg/ml antimycin
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A. The specific activity of complex III was calculated by subtracting the activity of the
104
nonenzymatic reaction from that of the total activity of complex III.
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The activity of complex IV was measured by monitoring the oxidation of reduced
106
cytochrome c at 550 nm. A 50 µM solution of reduced cytochrome c (92 to 97% reduced using
107
dithionite) in 10 mM KH2PO4, pH 7.0 was preincubated for 5 min. The reaction was initiated by
108
adding the homogenate (20 to 50 µg proteins) and the change in optical density was measured for
109
1.5 min.
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Mitochondrial isolations
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Gastrocnemius muscle and liver were removed, weighed and immediately placed in an ice-cold
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isolation medium consisting of 250 mM sucrose, 1 mM EGTA and 10 mM Tris/HCl, pH 7.4.
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Muscle mitochondria were isolated from gastrocnemius via an adaptation of the differential
115
centrifugation procedure, as used previously by Roussel et al. (2000). All steps were performed at
116
4°C.
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Muscles (2-2.5 g) were cut with scissors, minced using a Polytron (4 to 5 sec.) in an isolation
118
medium (20 ml/g tissue) then homogenized with a Potter-Elvehjem homogeniser (7 strokes). The
119
homogenate was centrifuged at 600 g for 10 min. The resulting supernatant was filtered through
120
cheesecloth and then centrifuged at 10 000 g for 10 min. The pellet was resuspended in the isolation
121
medium and then centrifuged at 10 000 g for 10 min. Finally, the pellet was resuspended in a
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minimal volume of respiratory medium consisting of 120 mM KCl, 5 mM KH2PO4, 3 mM
123
HEPES, 1 mM EGTA, 2 mM MgCl2 and 0.3 % (w/v) BSA, pH 7.4. Liver mitochondria were
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isolated by using a modification of the differential centrifugation procedure used previously by
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Krahenbuhl et al. (1994). The liver (6-7 g) was cut with scissors and homogenized using a Potter-
126
Elvehjem homogeniser (7 strokes) in the isolation medium (8 ml/g tissue). The homogenate was
127
centrifuged at 600 g for 10 min. The resulting supernatant was filtered through cheesecloth, then
128
centrifuged at 7 000 g for 10 min. The pellet was resuspended in the isolation medium (10 ml/g
129
tissue) and then centrifuged at 3 500 g for 10 min. The resulting pellet was resuspended in a minute
130
volume of respiratory medium. The lower g force of the last centrifugation step (3500 g) gives a
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more homogeneous mitochondrial preparation and is known to minimize the cytoplasmic
132
contamination and mitochondrial damage done to the pellet (< 10% in the present study) in addition
133
to maximizing the respiratory parameters (Goglia et al. 1988; Lanni et al. 1996).
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Mitochondrial respiration
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Oxygen was measured using a Clark oxygen electrode (oxygraph Hansatech), in a 2 ml glass cell,
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via continuous stirring at a constant temperature of 30°C. Mitochondria (0.4-0.6 mg protein/ml)
138
were incubated in the respiratory reaction medium as described above and saturated with room air.
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Substrate concentrations from liver measurements were 5 mM glutamate, 5 mM succinate and 5
140
mM ascorbate + 0.5 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). With regards to
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gastrocnemius measurements, 5 mM pyruvate + 5 mM malate, 5 mM succinate and 2 mM ascorbate
142
+ 0.5 mM TMPD were used. Inhibitor concentrations included 5 µM rotenone (to inhibit complex I
143
of the respiratory chain), and 3 mM myxothiazole (to inhibit complex III). The active state of
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respiration (state 3) was initiated by the addition of ADP (150 µM to the liver mitochondria or 200
145
µM to the gastrocnemius mitochondria). The basal non-phosphorylating respiration rate (state 4)
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was obtained by the addition of 3 µg/ml of oligomycin. RCR was the ratio of oxygen consumed
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after the addition of ADP to that consumed in the presence of oligomycin. The uncoupled state of
148
respiration was initiated by the addition of 2 µM of FCCP (carbonyl cyanide p-
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trifluoromethoxyphenylhydrazone). The respiratory parameters measured in isolated mitochondria
150
were normalized in relation to the specific activity of citrate synthase.
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Calculation of thermodynamic coupling and efficiency of oxidative phosphorylation
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Parameters were calculated using the methodology of Cairns et al (1998). The thermodynamic
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coupling of the energy conversion is designated by the dimensionless parameter q, known as the
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degree of coupling of oxidative phosphorylation.
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q = (1- (Jsh/Junc))1/2
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Jsh is the net oxygen consumption in state 4-oligomycin respiration, in the presence of oligomycin
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that inhibits ATP synthase. Junc is the uncoupled rate of oxygen uptake induced by the addition of
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FCCP, which dissipates the transmitochondrial proton gradient, and as a result ATP production
160
becomes nil.
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Kedem and Caplan (1965) have defined the efficiency of the energy conversion for oxidative
162
phosphorylation (η). Between state 4-oligomycin and state 3-uncoupled respiration, which represent
163
two steady states, an optimal thermodynamic efficiency of the energy conversion (ηopt) can be
164
discerned for any value of q (Stucki, 1980).
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ηopt = tan² (α/2), where α=arcsin q
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Oxidative phosphorylation should operate at a steady state for optimal efficiency for any given
167
degree of coupling. In addition, q can represent several well-defined values depending on the
168
energetic needs of the cell (Stucki, 1980). This theory is based on the thermodynamic trade-off of
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reducing efficiency to produce the maximum phosphate potential or increasing the efficiency to
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economize phosphate potential. Stucki (1980) has defined some physiological meanings for the
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degrees of mitochondrial oxidative coupling. The specific thermodynamic degrees of coupling
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correspond to the following set points with an unique maximal value of q: qecp (0.972) which is the
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economic net output power (phosphate potential) at optimal efficiency, qecf (0.953) being the
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economic net output flow (ATP), qp (0.910) as the maximal net output power and qf (0.786) as the
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maximal net output flow at optimal efficiency.
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In comparison to conventional measurements (RCR, ATP/O), non-equilibrium thermodynamics
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analysis provides a quantitative description and a better estimation of stoichiometry and the
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efficiency of energy conversion.
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Statistical analysis
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Results were expressed as mean ± standard deviation (SD). Means were compared by ANOVA
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using a Fisher Post-hoc test. A P value of < 0.05 was considered significant in all cases. All
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analyses were performed using StatView version 5.0 (SAS Institute, Cary, NC, USA).
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Results
Dexamethasone induced a significant reduction in food intake from day 2 (Fig. 1).
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Animals in the 3 groups did not differ, with respect to body weight, at the beginning of the
187
experimental procedure. Body weight decreased in DEX-treated rats from day 1, and PF animals
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from day 2 (Fig. 2). This decrease was significantly greater in DEX-treated rats than in PF animals,
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corresponding to 16% (DEX-treated) and 9% (PF) of initial body mass on the 4th day of treatment.
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At the same time, CON rats increased their body mass by 3.2% (Fig. 2).
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The overnight fasting decreased body weight in the 3 rats groups (Table 1). Liver weight
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was increased in DEX-treated animals by 23% vs. CON (P<0.01) and by 59% vs. PF (P<0.01)
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(Table 1). Conversely food restriction significantly decreased liver mass by 20% (PF rats vs. CON
194
rats). This difference in liver mass was not related to a change in hydration, as the dexamethasone-
195
treatment or the food restriction did not affect relative water contents (62.0 ± 3.0 vs. 66.0 ± 8.4 vs.
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62.1 ± 4.3 % per liver in DEX-treated, PF and CON rats). In DEX-treated rats, gastrocnemius mass
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was significantly decreased in comparaison with PF rats (-19%) and CON animals (-19%) (Table
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1). There was a significant increase in interscapular BAT mass in the DEX-treated group (+117%
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compared to PF and 90% compared to CON) (Table 1).
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In the liver, complex I activity was significantly higher in the DEX-treated group than in the
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PF group (+120%), but it was not different in comparison to the CON group (Table 2).
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Dexamethasone treatment significantly decreased the specific activity of complex IV (-28%
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compared to PF rats), although it was not different when compared to controls. There were no
204
significant difference in the specific activities of citrate synthase, succinate dehydrogenase and
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complex III between the DEX-treated and other groups (Table 2).
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In gastrocnemius, none of the enzymatic activities were significantly affected by
glucocorticoid treatment (Table 3).
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In the isolated liver mitochondria, the specific citrate synthase activity was significantly
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lower in the DEX-treated group than in the CON group (-28%) and PF group (-20%) (data not
210
shown). Basal non-phosphorylative respiration (state 4-oligomycin respiration) rates, normalized by
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the specific citrate synthase activity, are shown in Figure 3. With succinate and TMPD/ascorbate
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used as substrates, the state 4-oligomycin respiration was significantly increased (+46%) in isolated
213
liver mitochondria from DEX-treated rats compared to other groups. In the liver glutamate-respiring
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mitochondria, dexamethasone treatment significantly increased (+33%) the state 4-oligomycin
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oxygen consumption in comparison to PF rats, but not when compared to CON rats.
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Table 4 shows that the respiratory parameters in the liver, expressed per mg of mitochondria
protein, were similar, across groups regardless of the respiratory substrate.
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In gastrocnemius none of the respiratory parameters were affected by dexamethasone
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treatment whatever the substrate used (Table 5). When gastrocnemius respiratory parameters were
220
normalized by the CS activity, no difference was observed between groups (data not shown).
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In liver succinate-respiring mitochondria, q was significantly decreased by dexamethasone
222
treatment. With glutamate, q was marginally (P=0.12) lower in DEX-treated and PF than in CON
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rats (Table 6). Similar results were obtained for the determined thermodynamic optimal efficiency
224
of oxidative phosphorylation (ηopt).
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In gastrocnemius, q and ηopt were similar in the 3 rats groups (Table 7).
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Discussion
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The present study reports that the induction of a catabolic state, by dexamethasone, results in an
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increased liver mass and increased non-phosphorylative oxygen consumption in liver mitochondria.
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In addition, we found a decreased thermodynamic coupling and efficiency of the oxidative
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phosphorylation in the complex I and II respiratory pathways in the liver mitochondria of DEX-
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treated rats. In contrast, dexamethasone induced gastrocnemius mass atrophy without affecting
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mitochondrial energy metabolism.
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While the citrate-synthase activity was not modified in the 600 g homogenate it was
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decreased in isolated mitochondria of liver in DEX-treated rats. Moreover, we found a lower
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percentage of intact mitochondria in isolated liver mitochondria of DEX-treated rats (91%
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compared to 96% and 94% in CON and PF groups). Therefore, for the measurement of parameters
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in isolated mitochondria preparations, we used the specific citrate synthase activity as a
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mitochondrial marker enzyme. These parameters/CS ratios may reflect a change originating from
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the mitochondria themselves, rather than from the homogeneization or measurement procedures.
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Of particular interest is the finding that dexamethasone treatment significantly increased
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liver non-phosphorylative oxygen consumption using succinate (+46%) and ascorbate as substrates
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(+46%; versus CON and PF). With regard to glutamate, state 4-oligomycin respiration was not
243
different compared to control rats, but it was higher (+33%) in DEX-treated relative to PF animals.
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Therefore, it appears that there is a specific catabolic state-related increase (+42%) in the non-
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phosphorylative oxygen consumption of liver mitochondria. Indeed, state 4-oligomycin respiration
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was increased in DEX-treated rats compared to CON rats, except in complex I (effect of food
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restriction and/or hypercatabolism), and consistent increases were found when DEX-treated animals
248
were compared to PF animals (effect of hypercatabolism). On the other hand, food restriction
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appears to reduce state 4-oligomycin oxygen consumption (via complex I) in PF compared to CON
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rats. Such a change in these non-phosphorylative conditions could be due to modifications in
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respiratory chain activity, inner membrane conductance (proton leak) or the intrinsic coupling of the
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respiratory chain (H+/2e-). The present acute treatment using dexamethasone did not modify
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respiratory chain complex activities in the liver. As a result, this strongly suggests that basal
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proton conductance and/or the efficiency at the level of the respiratory chain did change in the liver
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mitochondria. In the latter case, we can speculate an effect located on the cytochrome oxidase as
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state 4-oligomycin respiration by using ascorbate/TMPD as substrate was increased. Moreover, it
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has been demonstrated that cytochrome oxidase H+/2e- stoichiometry is variable and represents a
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possible location for intrinsic uncoupling at the level of the respiratory chain (Capitanio et al. 1991;
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Papa et al. 1991; Piquet et al. 2000). However, further experiments are necessary to confirm these
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possibilities. Whatever the mechanism involved, this represents a substantial increase in state 4
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energy wastage. Moreover, it is interesting to note that proton leak and redox slipping may affect
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the oxidative phosphorylation yield. Our results agree with data obtained from the long-term
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administration (3-7 days) of high-dose glucocorticoids, which are reported to decrease liver
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oxidative phosphorylation (state 3 respiration) via complex I respiratory substrates while remaining
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unchanged when fuelled through complex II or IV (Kerppola, 1960; Kimura & Rasmussen, 1977;
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Jani et al. 1991). Our findings that state 4 respiration is increased are therefore complementary to
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those studies since it has never been studied in DEX-treated rats, although inconsistencies were
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shown in corticosterone-treated rats (Jani et al. 1991).
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Mitochondria can vary the efficiency of oxidative phosphorylation in order to respond to one
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of four physiological missions: 1) maximizing ATP production with a corresponding value qf for
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the thermodynamic degree of coupling, 2) maximizing the cellular phosphate potential (qp), 3)
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minimizing the cost of production (qecf ) and 4) a combination of all three (qecp) (Stucki JW, 1980).
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In the control animals used in our study, the experimentally derived q value for the complex I and II
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pathways were between that of qp and qecf. Therefore, the liver mitochondria of the control animals
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adapted their function for the economic production of ATP in addition to maintaining the phosphate
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potential. Dexamethasone decreases the degree of thermodynamic coupling of oxidative
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phosphorylation in both complex I and II respiratory pathways. Their values were between qf and qp
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and nearly that of qp. In relation to this result, the thermodynamic optimal efficiency of energy
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conversion (ηopt) was also decreased by dexamethasone. These results therefore suggest that in
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DEX-treated rats, liver mitochondria adapt their function for maximum ATP production and also
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to maintain cellular phosphate potential at the expense of the energy conversion efficiency. Similar
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adaptive reductions in the efficiency of oxidative phosphorylation has already been noted by
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Nogueira et al (2001) who showed that cellular respiratory rate increases in liver mitochondria of
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either hyperthyroid rats or animals exposed to a polyunsatured fatty acid deficient diet.
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It is well known that liver mass is decreased during energy restriction, which in turn could
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partly explain why energy expenditure falls in response to a reduced food intake (Ramsey et al.
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2000). In the present study, liver weight was decreased by food restriction (PF versus control rats)
288
and higher (23 to 59%) in DEX-treated rats compared with other rat groups. Such an increase in
289
liver mass could result from an increased glycogen content (Weber & Kletzien, 1982; Michaels &
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Cardell, 1997; Bollen et al. 1998) although we did not find any difference in hydratation.
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Alternatively, hepatic lipid content is increased in DEX-treated rats, but is insufficient to fully
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explain the increased liver mass (Kaur N et al. 1989; Palacios et al. 1995; Franco-Colin et al. 2000).
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Finally, dexamethasone treatment increase liver protein synthesis and therefore metabolic tissue
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(Odedra et al. 1983; Savary et al. 2001). This suggests that both maintenance and growth costs may
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be increased.
296
Our study demonstrated that a dexamethasone-induced hypercatabolic state results in
297
skeletal muscle atrophy without any change in mitochondrial energy metabolism. Indeed, none of
298
the respiratory complex activities, oxygen consumption rates or the thermodynamic degree of
299
coupling of oxidative phosphorylation, were altered in the mitochondria of DEX-treated rats. This is
300
in agreement with previously reported effects of glucocorticoid treatment (Vignos & Greene, 1973;
301
Capaccio et al. 1985; Marone et al, 1994). These results suggest that the mitochondrial metabolism
302
of skeletal muscle produces enough ATP to fulfil either the cellular energy requirement and/or the
303
energy-dependent pathways induced by glucocorticoids, such as the energy-ubiquin-dependent
304
proteolytic pathway (Tiao G et al. 1996; Mitch et al. 1999), and the energy-dependent glutamine
305
synthase activity pathway (Max SR et al, 1988; Minet-Quinard et al. 1999, 2000).
16
306
The main thermogenic tissue in rats is brown adipose tissue, the weight of which is largely
HAL author manuscript
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307
increased (+100%, present study) by glucocorticoid injection. Previous studies have clearly
308
demonstrated that such an increase in the BAT mass was due to increased lipid storage rather than
309
an increased thermogenic capacity of this tissue (Mazzuccheli et al. 1960; Strack et al. 1995).
310
Furthermore, glucocorticoids are known to reduce the activity or the gene expression of UCP1
311
(Tokuyama & Himms-Hagen, 1989; Moriscot et al. 1993; Strack et al. 1995). Moreover, in our
312
study we found no effect of dexamethasone on mitochondrial oxidative capacity (cytochrome c
313
oxidase activity; data not shown). Therefore, it is unlikely that interscapular BAT increases energy
314
expenditure in DEX-treated rats.
315
A reduction in body size (a 16% weight loss in the present study) generally results in
316
lowering of energy expenditure per whole rat, because of the reduced maintenance requirement
317
(Ramsey et al. 2000). In the present study, the dexamethasone-related decrease in the body weight
318
was greater than that observed in the PF rats, highlighting an increased whole body energy
319
expenditure. This is paradoxical in view of the reducing effect of food restriction on energy
320
expenditure (Ramsey et al. 2000). Furthermore, in our study, dexamethasone treatment increased
321
the liver-to-body weight ratio while the relative skeletal muscle mass remained unchanged. Since
322
liver and muscle are the main contributors to standard metabolic rate in rats (Rolfe & Brown, 1997),
323
it is likely that the liver would effectively contribute to the increased energy expenditure despite the
324
body weight loss (Woodward & Emery, 1989). Indeed, if we assume that liver contributes 20% to
325
the metabolic rate of a rat (Rolfe & Brown, 1997), then the 60-80% gain in the liver-to-body weight
326
ratio reported herein, could increase total energy expenditure by 12-16%.
327
administered in humans and rats results in an increased energy expenditure by 10-20% (Woodward
328
& Emery, 1989; Brillon et al. 1995; Tataranni et al. 1996). Obviously, we can not rule out the
329
influence of other biochemical mechanisms or possible determinants of energy balance which have
330
accounted also for the weight loss.
Glucocorticoids
331
In conclusion, 5 days of high-dose dexamethasone treatment induced a significant increase
332
in liver mass, an increase in liver mitochondrial non-phosphorylative oxygen consumption rate from
17
HAL author manuscript
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333
all substrates used, and a decrease in the thermodynamic coupling of oxidative phosphorylation in
334
liver respiratory pathways. We suggest therefore that dexamethasone increases proton leak and/or
335
redox slipping in liver mitochondria, which in turn is probably responsible for the decrease in the
336
thermodynamic efficiency of energy conversion. Thus, rats would adapt their mitochondrial energy
337
functions to a dexamethasone-induced hypermetabolic state by maximizing ATP production in
338
addition to maintaining their cellular energy state, regardless of the cost. This treatment has no
339
effect on energy metabolism in the muscle. Together with a decreased food intake, the increase in
340
energy expenditure induced by high dose of dexamethasone results in a negative energy balance and
341
thus weight loss.
18
Acknowledgements
Authors thank Pierre Legras, Jérôme Roux and Dominique Gilbert for animal care, Antoine
Augeraud for technical assistance and to Miriam Ryan for her assistance in correcting the
HAL author manuscript
english. This work was supported by grants from Contrat de Plan Etat Region 2000-2004.
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19
Textes footnotes
BAT, brown adipose tissue; CS, citrate synthase; DCPIP, 2,3-dichlorophenollindophenol;
FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PMS, phenazine methosulfate;
HAL author manuscript
RCR, respiratory control ratio; TCA, tricarboxylic acid; TMPD, N,N,N',N'-tetramethyl-pphenylenediamine; TNB, thio-bis(2-nitrobenzoic acid).
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20
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27
Figure 1. Daily food intake during the last 4 days of treatment, for control (CON) (▲) and dexamethasone
(DEX)-treated (♦) rats. For details of procedure see Materials and methods section. Values expressed as the
mean for eight rats per group (standard deviation denoted by vertical bars). Significant differences indicated:
HAL author manuscript
† P < 0.01 vs. CON.
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28
Figure 2. Body weight changes during the first 4 days of treatment for pair-fed (PF) (■), control (CON)
(▲) and dexamethasone (DEX)-treated (♦) rats. For details of procedure see Materials and methods section.
Values expressed as the mean for eight rats per group (standard deviation denoted by vertical bars).
HAL author manuscript
Significant differences indicated: † P < 0.01 vs. CON; ** P < 0.05 vs. PF.
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29
Figure 3. State 4-oligomycin respiration normalized by the specific citrate synthase activity in liver
isolated mitochondria from pair-fed (PF), control (CON) and dexamethasone (DEX)-treated rats. For details
of procedure see Materials and methods section. Values expressed as the mean for eight rats per group
HAL author manuscript
(standard deviation denoted by vertical bars). Significant differences indicated: ‡ P < 0.05 vs. CON; ** P <
0.05 vs. PF.
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30
Table 1. Body weight and organ mass: liver, gastrocnemius muscle and interscapular brown adipose tissue
in pair-fed (PF), control (CON) and dexamethasone (DEX)-treated rats.
(Values expressed as mean ± standard deviation for eight rats per group)
HAL author manuscript
inserm-00128557, version 1
DEX-treated
PF
CON
ANOVA
310 ± 28
329 ± 38
329 ± 32
NS
242 ± 19 †*
285 ± 37
315 ± 33
P = 0.0006
13.38 ± 2.30 †*
8.58 ± 1.12 †
10.84 ± 1.47
P < 0.0001
5.49 ± 0.66 †*
3.04 ± 0.28
3.44 ± 0.24
P < 0.0001
3.27 ± 0.21 †*
4.04 ± 0.50
4.25 ± 0.27
P < 0.0001
g/100g body weight
1.36 ± 0.07
1.41 ± 0.06
1.36 ± 0.09
NS
Brown adipose tissue, g
0.76 ± 0.20 †*
0.35 ± 0.10
0.40 ± 0,14
P < 0.0001
g/100g body weight
0.31 ± 0.08 †*
0.12 ± 0.04
0.12 ± 0.04
P < 0.0001
Initial body weight, g
Sacrifice body weight, g
Liver, g
g/100g body weight
Gastrocnemius, g
Significant differences indicated: * P < 0.01 vs. PF, † P < 0.01 vs. CON; NS: not significant.
For details of procedure see Materials and methods.
31
Table 2. Mitochondrial enzyme activities in liver 600 g homogenate from pair-fed (PF), control (CON)
and dexamethasone (DEX)-treated rats.
(Values expressed as mean ± standard deviation for eight rats per group)
HAL author manuscript
Enzymatic activity
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DEX-treated
PF
CON
ANOVA
Citrate Synthase
151 ± 27
158 ± 23
152 ± 26
NS
Succinate Dehydrogenase
101 ± 26
107 ± 30
109 ± 26
NS
NADH-ubiquinone reductase
44 ± 11 *
20 ± 8 †
44 ± 20
P < 0.01
88 ± 57
86 ± 52
101 ± 81
NS
91 ± 31 **
126 ± 29
100 ± 21
P < 0.05
(nmol/min per mg of protein)
(Complex I)
Ubiquinol-cytochrome c
reductase (Complex III)
Cytochrome c oxidase
(Complex IV)
Significant differences indicated: * P < 0.01 vs. PF, † P < 0.01 vs. CON; ** P < 0.05 vs. PF; NS: not
significant.
For details of procedures see materials and methods section.
32
Table 3. Mitochondrial enzyme activities in gastrocnemius muscle 600 g homogenate from pair-fed (PF),
control (CON) and dexamethasone (DEX)-treated rats.
(Mean values with their standard deviation for eight rats per group)
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Enzymatic activity
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DEX-treated
PF
CON
ANOVA
Citrate Synthase
602 ± 239
653 ± 314
538 ± 183
NS
Succinate Dehydrogenase
130 ± 65
147 ± 63
125 ± 49
NS
NADH-ubiquinone reductase
111 ± 57
99 ± 76
85 ± 32
NS
382 ± 190
363 ± 208
409 ± 170
NS
166 ± 66
213 ± 112
194 ± 84
NS
(nmol/min per mg of protein)
(Complex I)
Ubiquinol-cytochrome c
reductase (Complex III)
Cytochrome c oxidase
(Complex IV)
NS: not significant.
For details of procedures see materials and methods section.
33
Table 4. Respiratory parameters of liver isolated mitochondria from pair-fed (PF), control (CON) and
dexamethasone (DEX)-treated rats.
HAL author manuscript
(Values expressed as mean ± standard deviation for eight rats per group)
Respiratory substrates
Parameters
Glutamate
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Succinate
TMPD/ascorbate
DEX-treated
PF
CON
ANOVA
State 3
35 ± 16
26 ± 13
43 ± 19
NS
State 4
5±1
5±1
6±1
NS
RCR
7±3
5±3
7±3
NS
Uncoupled state
29 ± 17
24 ± 13
46 ± 24
NS
State 3
76 ± 29
79 ± 28
98 ± 16
NS
State 4
18 ± 5
14 ± 3
18 ± 2
NS
RCR
4.2 ± 1.4
5.6 ± 1.4
5.4 ± 0.8
NS
Uncoupled state
93 ± 26
103 ± 31
121 ± 21
NS
State 3
131 ± 34
122 ± 13
147 ± 27
NS
State 4
98 ± 26
81 ± 16
96 ± 16
NS
RCR
1.3 ± 0.1
1.5 ± 0.2
1.5 ± 0.1
NS
Uncoupled state
167 ± 39
163 ± 30
190 ± 41
NS
NS: not significant.
State 3, state 4 and uncoupled state respiration expressed as natomes of oxygen/min per mg
mitochondrial protein. For details of procedures see materials and methods section.
34
Table 5. Respiratory parameters of gastrocnemius isolated mitochondria from pair-fed (PF), control
(CON) and dexamethasone (DEX)-treated rats.
HAL author manuscript
(Values expressed as mean ± standard deviation for eight rats per group)
Respiratory substrates
Parameters
inserm-00128557, version 1
DEX-treated
PF
CON
ANOVA
Pyruvate + malate
State 3
70 ± 39
105 ± 48
85 ± 30
NS
State 4
8±4
9±2
7±2
NS
9±
11.7 ±
12.8 ±
NS
Uncoupled state
123 ± 74
174 ± 37
133 ± 41
NS
State 3
88 ± 29
118 ± 26
120 ± 44
NS
State 4
24 ± 13
35 ± 21
27 ± 16
NS
3.7 ±
3.4 ±
4.4 ±
NS
Uncoupled state
123 ± 46
156 ± 53
128 ± 59
NS
State 3
260 ± 117
289 ± 75
231 ± 61
NS
State 4
172 ± 84
186 ± 58
156 ± 39
NS
RCR
1.5 ± 0.2
1.5 ± 0.2
1.5 ± 0.3
NS
Uncoupled state
385 ± 128
354 ± 108
403 ± 121
NS
RCR
Succinate
RCR
TMPD/ascorbate
NS: not significant.
State 3, state 4 and uncoupled state respiration expressed as natomes of oxygen/min per mg
mitochondrial protein. For details of procedures see materials and methods section.
35
Table 6. Thermodynamic degree of coupling (q ) and optimal efficiency (η
ηopt ) of the oxidative
phosphorylation in liver isolated mitochondria from pair-fed (PF), control (CON) and dexamethasone
(DEX)-treated rats.
HAL author manuscript
(Values expressed as mean ± standard deviation for eight rats per group)
Respiratory substrates
Glutamate
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Succinate
Ascorbate/TMPD
Parameters
DEX-treated
PF
CON
ANOVA
q
0.903 ± 0.044
0.896 ± 0.040
0.942 ± 0.013
NS
ηopt
0.413 ± 0.092
0.396 ± 0.080
0.500 ± 0.039
NS
q
0.896 ± 0.019 ‡**
0.923 ± 0.022
0.922 ± 0.011
P < 0.05
ηopt
0.387 ± 0.038 ‡**
0.451 ± 0.052
0.439 ± 0.028
P < 0.05
q
0.656 ± 0.028 †*
0.708 ± 0.024
0.706 ± 0.028
P < 0.005
ηopt
0.140 ± 0.016 †*
0.173 ± 0.016
0.173 ± 0.018
P < 0.005
Significant differences indicated: ** P < 0.05 vs. PF; ‡ P < 0.05 vs. CON; * P < 0.01 vs. PF; † P <
0.01 vs. CON. NS: not significant.
For details of procedures see materials and methods section.
36
Table 7. Thermodynamic degree of coupling (q ) and optimal efficiency (η
ηopt ) of the oxidative
phosphorylation in gastrocnemius isolated mitochondria from pair-fed (PF), control (CON) and
dexamethasone (DEX)-treated rats.
HAL author manuscript
(Values expressed as mean ± standard deviation for eight rats per group)
inserm-00128557, version 1
Respiratory substrates
Parameters
DEX-treated
PF
CON
ANOVA
Pyruvate + malate
q
0.966 ± 0.011
0.972 ± 0.016
0.972 ± 0.011
NS
ηopt
0.593 ± 0.059
0.635 ± 0.089
0.628 ± 0.059
NS
q
0.889 ± 0.039
0.880 ± 0.052
0.887 ± 0.036
NS
ηopt
0.400 ± 0.076
0.374 ± 0.105
0.377 ± 0.064
NS
q
0.749 ± 0.073
0.713 ± 0.025
0.774 ± 0.050
NS
ηopt
0.214 ± 0.083
0.176 ± 0.017
0.229 ± 0.051
NS
Succinate
Ascorbate/TMPD
NS: not significant.
For details of procedures see materials and methods section.
food intake (g)
0
5
10
15
20
25
30
day 1
day 2
†
day 3
†
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35
day 4
†
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37
75
80
85
90
95
100
day 0
day 1
†**
day 2
†**
†
day 3
†**
†
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relative body weight (%)
105
day 4
†**
†
HAL author manuscript
38
0
5
10
15
20
25
a
glutamate
b
a, b
0
10
20
30
40
50
60
70
80
90
100
a
succinate
b
b
0
100
200
300
400
500
600
inserm-00128557, version 1
State 4-oligomycine respiration
(10e-3 natomes O/nmoles)
a
ascorbate/TMPD
b
CON
PF
b
DEX-treated
HAL author manuscript
39